1. Field of the Invention
The present disclosure relates to a radial-shaped macromolecule containing iodine as an active ingredient for the computed tomography (CT) contrast medium, a method of preparing the same, and a CT contrast medium composition including the radial-shaped macromolecule.
2. Description of the Related Art
Computed tomography (CT) denotes a photographic technique in which a target area of the human body was irradiated with X-ray in various directions and the transmitted X-ray is collected with a detector, and then the difference between X-ray absorptions with respect to the area is reconstructed through a mathematical technique by using a computer. That is, it is an imaging technique producing a three-dimensional image through computer programming by recombining many two-dimensional X-ray photographs taken at various different angles with a camera rotating 360 degrees around the body.
Therefore, such X-ray-based imaging generally generates images mainly focused on bones. If the CT imaging is performed after the injection of a contrast medium based on a relatively high atomic number such as barium or iodine into the blood vessel of a living animal or human, X-ray cannot transmit clearly through the area where such contrast medium exists due to photoelectric effect, but will be absorbed to exhibit white or light grayish color on the produced images similar to the bones which is shown in very bright white color. In general, the degree (α) of absorption of X-ray is proportional to the atomic number (N) and the wavelength (λ) of the X-ray. That is, the degree (α) of absorption of X-ray is inversely proportional to the energy of X-ray.α=N5λ7/2 
CT along with magnetic resonance imaging (MRI) provides anatomical information, while PET (positron emission tomography) and SPECT (single photon emission computed tomography) imaging techniques provide information on physiological/biochemical functions. Also, despite the lower resolution of CT compared to that of MRI, CT is indispensable in clinic because of its short scan time, relatively low price for a CT scan, and good accessibility to the CT equipment as most of the hospitals own it. That is, because the total CT scan time is about one-tenth or less of the MRI scan time, CT imaging is highly indispensable particularly for emergency patients with critical brain injury requiring rapid diagnosis. Also, the patient discomfort during the MRI scan due to the noise and long scan time can be reduced by CT scan, the access to the CT equipment is facile, and the cost for a CT scan is about one-tenth of that of MRI scan.
To date, small molecular CT contrast media containing iodine used in clinic are broadly classified into two types: ionic and non-ionic compounds.

Because the severity of side effects by the non-ionic compounds are much less than that of the ionic compounds, non-ionic CT contrast media are mostly administered these days to the patients. Also, two or three phenyl rings in the conventional small molecular non-ionic CT contrast media compounds containing iodine were linked by covalent bonds to extend the in vivo circulation time by increasing the molecular weight. However, high-dose amount for up to 80-90 g of these small molecular CT contrast media containing iodine need to be administered to adults depending on their weight in order to obtain a proper level of CT signals because of their short in vivo circulation time (half-life). Thus, adverse effects such as allergy and shock are exhibited occasionally in some patients, and in the limited worst case, life may be threatened. Such events occur more frequently when attaining sufficient circulation time is necessary by administrating high-dose amount of contrast medium for effective diagnosis of cardiovascular diseases such as the heart area. Therefore, in order to overcome these problems, there is an urgent need to develop a safer compound which can produce excellent contrast enhancement for a prolonged period by administrating relatively low-dose amount of CT contrast media. In addition, other limitations of the current small molecular CT contrast media containing iodine may include low LD50 value, high osmolality, high viscosity, etc.
In order to overcome these limitations of small molecular CT contrast media containing iodine, the development of new macromolecular CT contrast media containing iodine has been reported recently. For example, multiple units of (commercially available) small molecular compounds containing iodine or their derivatives have been either covalently attached to the macromolecular scaffolds or physically encapsulated inside the macromolecular assemblies such as liposomes, micelles, and other nanoparticles.
Methods of encapsulating commercially available small molecular CT contrast media containing iodine or their derivatives in the macromolecular liposomes were disclosed in the Non-Patent Literatures 1 [J. Zheng, et al., Mol. Pharmaceutics 2009, 6, 571-580], 2 [A. Sachse, et al., Invest. Radiol. 1997, 32, 44-50], 3 [E. Samei, et al., Int. J. Nanomedicine 2009, 4, 277-282], and 4 [C. Y. Kao, et al., Acad. Radiol. 2003, 10, 475-483].
Methods of encapsulating commercially available small molecular CT contrast media containing iodine or their derivatives in the macromolecular micelles were disclosed in the Non-Patent Literatures 5 [V. S. Trubetskoy, et al., J. Drug Target. 1997, 4, 381-388] and 6 [V. P. Torchilin et al., Acad. Radiol. 1999, 6, 61-65]
A method of encapsulating commercially available small molecular CT contrast media containing iodine or their derivatives in the macromolecular nanoparticles was disclosed in the Non-Patent Literature 7 [F. Hyafil, et al., Nat. Med. 2007, 13, 636-641].
Regarding the Non-Patent Literature 7, the carboxylic acid end group of the iodinated compound was capped with an alkyl group to enhance the hydrophobicity of the compound, which facilitated the internalization of the iodinated compound into the interior of the nanoparticle. When these iodine-containing nanoparticles were injected into the blood vessels, generally they were readily taken up by macrophages and accumulated increasingly in the areas of inflammation, cancer, and arteriosclerosis with time where relatively larger amount of macrophages exists, to facilitate the diagnosis of related diseases by CT imaging. Meanwhile, regarding the Non-Patent Literature 5, the micellar structures were formed from the hydrophilic poly(ethylene glycol) (PEG) with its one end substituted with a short peptide containing several units of small molecular hydrophobic iodinated compounds, to extend in vivo circulation time. However, the structures of these liposomes and micelles based on self-assembly are not intact and can be easily disassembled by the changes in the surrounding media in vivo to release the entire or a portion of toxic iodinated small molecular contrast agents.
Therefore, the development of CT contrast media based on metal nanoparticles that can exhibit contrast enhancement by themselves without the need for the encapsulation of small molecular compounds containing iodine have been reported.
A CT contrast medium based on gold (Au) nanoparticles with PEG groups substituted on the surface was disclosed in the Patent Literature 1 [PCT/KR2006/003452].
A CT contrast medium based on gold nanoparticles coated with heparin was disclosed in the Non-Patent Literature 8 [I. C., Sun, et al., Chem. Eur. J. 2009, 15, 13341-13347].
A CT contrast medium based on uniformly sized tantalum (Ta) oxide nanoparticles substituted with PEG and fluorophores was disclosed in the Non-Patent Literature 9 [M. H. Oh, et al., J. Am. Chem. Soc. 2011, 133, 5508-5515].
For the CT contrast media based on metal nanoparticles, the metal (Au, Ta, etc.) itself serves as an imaging agent (i.e., absorbs X-ray beam) which was coated with biocompatible PEG and heparin to extend the in vivo circulation time and to facilitate imaging of specific organs through the accumulation in the liver, respectively.
However, the foregoing macromolecular contrast media are expected to have problems such as high costs and toxicity due to the limited excretion and long-term in vivo accumulation (i.e., half-lives of more than years to decades). Also difficulties are expected for these macromolecules in maintaining structural integrity in vivo (e.g., self-assembled structures) and achieving reproducibility in manufacturing to obtain macromolecular mixtures of the same molecular weight distribution to elicit same effects. Also, more difficulties are expected in commercializing these materials based on macromolecular mixtures for human administration through clinical testing due to the lack of standardized global protocols for toxicity evaluations.
Meanwhile, examples of liposome-based vascular contrast media for CT with encapsulated small molecular compounds containing iodine are Fenestra (ART Advanced Research Technologies Inc., Canada) and eXIA160 (Binitio Biomedical, Inc., Canada), which are both sold for animal applications only. In fact there is more pressure on the researchers in the field of basic medical research using micro-CT, not only because the price for one vial (2.5 mL) of these contrast media permitting 5-10 injections into the blood vessels of mice or rats is about one million wons, but also the injection of these contrast media into the tail vein of small animals are not easy. On the other hand, if a small molecular contrast medium for human is injected instead into the small animals, achieving a proper level of contrast enhancement is impossible because most of the contrast agents is already discharged into the bladder through kidney during the first one to three minutes of the preparation period after the injection, which involves transferring the animal to the imaging bed and setting the parameters, such as scan range, in the operating software before initiating the CT scan. Therefore, there is an urgent need to develop a vascular contrast medium for CT which is inexpensive and well-excreted after relatively prolonged in vivo circulation without the need for high-dose amount, not only for the diagnosis of cardiovascular diseases requiring high-dose amount, but also for the micro-CT experiments using small animals.
For this purpose, papers and patents related to CT contrast media containing iodine using dendrimers as macromolecular scaffolds started to appear around 1990. That is, if a monomolecular dendrimer is used as a macromolecular scaffold (see descriptions below), the reproducibility in terms of both the preparation and effects can be achieved. Also, the facile excretion after certain period of intravascular circulation is expected, because the size of the dendrimers is much smaller than that of the contrast agents based on metal nanoparticles or self-assembly of linear polymers.
The dendrimer is a relatively small treelike (or radial-shaped) macromolecule of generally 10 nm or less in diameter, and is a pure molecule with a single molecular weight value made by stepwise (i.e., adding one layer) iterative organic synthesis and purification. Unlike most of other traditional macromolecules (polydispersed mixtures), dendrimers have predictable size in a specific environment (solvent, pH, temperature, etc.) and thus are advantageous for the applications which require the use of macromolecules of specific hydrodynamic diameters. Some advantages of dendrimers include structural integrity, unlimited possibility to vary the component functional groups by organic synthesis and the corresponding physicochemical properties, feasibility to covalently attach various functional units (e.g., small molecular drugs, targeting agents, surface modifiers, etc.) to the interior and the surface of the dendrimers, and very low enzymatic degradation rate which is important for biological applications.
Examples of using dendrimers for biomedical applications include, gene transfection by forming charge complex between the polycationic dendrimers and anionic genes; drug delivery where the drugs were either encapsulated in the interior void of the dendrimers or covalently attached to the dendrimers so they can be released by a specific stimulus (pH, light, enzyme, etc.) at the disease sites; targeted delivery or controlled release of drugs by modifying the structures of carrier dendrimers; multivalent effects through which the binding affinity between the carbohydrate ligands and lectins at the extracellular membrane can be enhanced significantly; medical diagnosis for the signal amplification of imaging agents by substituting multiple copies of small molecular imaging agents at the dendrimer scaffold; tissue engineering using biocompatible and biodegradable dendrimers.
Since the advent of the first dendrimer in the late 1970s by Denkewalter as a polylysine dendrimer, the research focus on the dendrimer field has shifted from the molecular design of new dendrimers and development of their synthetic methodologies to the investigation of the physicochemical properties of various types of dendrimers and some basic applications utilizing their specific properties (e.g., self-assembly, biomimetic systems, etc.), and more recently, to the advanced applications of dendrimers for materials science and biomedicine. An example of the structure of the polylysine dendrimer synthesized by Denkewalter is shown below (Patent Literature 2 [U.S. Pat. No. 4,410,688]).

Also, the poly(amidoamine) (PAMAM) dendrimer was developed by Dr. Donald A. Tomalia in the 1980s while he was working at the Dow Chemical company. The interior of PAMAM dendrimer is composed of aliphatic amino and amide groups and the surface groups can be amine, carboxylate, hydroxyl, and so on. For example, the structures of the first, second, third, and fourth generation (G1, G2, G3, and G4) PAMAM dendrimers with ethylenediamine as a core unit and the amine as the surface group are shown below.

PAMAM dendrimers are popular for in vivo applications among several commercially available dendrimers, and have been widely used for various biomedical applications. While the dendrimers made by stepwise organic synthesis and purification are, in principle, monomolecular with single molecular weight values, these commercially available PAMAM dendrimers (manufactured by Dendritech, Inc., sold by Sigma-Aldrich Corporation) contain structural defects and are heterogeneous (polydispersity index (PDI), ca. 1.01-1.1) with some unwanted side-products because they were synthesized through a divergent approach where the excess amount of reagents were added for reactions and the purification was done roughly for large-scale production. Thus, the structural analysis of PAMAM dendrimers or their derivatives at the monomolecular level with a single molecular weight is not possible. However, it would be reasonable to use PAMAM dendrimers in order to quickly examine the potential of adopting dendrimers for specific biological applications before designing and synthesizing an optimal biocompatible dendrimer with a single molecular weight value.
Not many examples of dendrimer-based CT contrast media containing iodine have been reported to date. This could be due to the complex preparation methods involving many synthetic steps, relatively high toxicity, minor degree of extension of the duration of contrast enhancement compared to that of small molecular contrast agents containing iodine, and relatively poor image quality (i.e., contrast enhancement) (Non-Patent Literature 10 [W. Krause, et al., Top. Curr. Chem. 2000, 210, 261-308], Patent Literature 3 [PCT/EP94/04245], Patent Literature 4 [PCT/EP96/02450], Patent Literature 5 [PCT/FR92/01135], Patent Literature 6 [PCT/EP94/648203], Patent Literature 7 [PCT/EP95/730573], Patent Literature 8 [PCT/EP95/78263], Non-Patent Literature 11 [A. T. Yordanov, et al., Nano Lett. 2002, 2, 595-599], and Non-Patent Literature 12 [Y. Fu, et al., Bioconjugate Chem. 2006, 17, 1043-1056]).
Meanwhile, a greater amount of research has been conducted on the dendrimer-based MRI contrast media, for example, Bayer Schering Pharma AG. who developed gadopentetate dimeglumine (Gd-DTPA, brand name: Magnevist), a representative small molecular vascular contrast medium for MRI currently administered to patients in the hospital, has developed the dendrimer-based vascular contrast medium for MRI (EP 430863, WO 97/02051, WO 98/24775, WO 98/24774, and U.S. Pat. No. 5,911,971), and the clinical trials for “Gadomer-17” (or referred to as “Gd-DTPA-17” or “SH L643 A”; K. Nael, et al., J. Magn. Reson. Imaging 2007, 25, 66-72; B. Misselwitz, et al., Magn. Reson. Mater. Phys. Biol. Med. 2001, 12, 128-134) are underway.
Therefore, there is a current need to develop a CT contrast medium including iodine-containing radial-shaped macromolecules with excellent contrast enhancement, which can overcome the current limitations of the small molecular CT contrast agents containing iodine used in clinic. Ideally, this CT contrast medium should be made by relatively simple synthetic methods, be inexpensive, have prolonged intravascular circulation time sufficient for the diagnosis of cardiovascular diseases, and be mostly excreted through a safe route at the adequate time point after the administration.
Accordingly, while conducting research on the CT contrast medium that can overcome foregoing limitations, the present inventors confirmed that by using the iodine-containing radial-shaped macromolecules featuring a protective layer made of biocompatible polymers which surrounds the iodine-containing compounds substituted in the core or the interior region, the duration of contrast enhancement has been significantly improved in comparison to that of the current small molecular contrast media compounds containing iodine, the in vivo toxicity has been reduced by forming a protective layer with relatively long biocompatible polymer chains which prevents the exposure of the toxic iodine-containing compounds substituted near the surface of the core region to the external environment for potential adverse effects, the in vivo circulation time has been increased by preventing rapid uptake by macrophages, the excretion has been realized at the adequate time point after the intravascular injection, the large-scale production at high yield and low cost is amenable due to the simple preparation and purification methods, the reproducibility in terms of the preparation and effects is high due to the relatively low polydispersity, and the structural integrity of the iodine-containing radial-shaped macromolecules is ensured because the compound is entirely formed by covalent binds, thereby leading to the completion of the present invention.